With one exception, all of the major proteins of the human red cell membrane have been purified and characterized in great detail. The exception is the protein known as band 4.2 which comprises approximately 5% of the total membrane protein and is present in a number which equals or exceeds that of the major membrane skeletal proteins such as spectrin and band 4.1. Although early studies suggested that this protein is tetrameric and may bind to band 3, almost nothing is known about its properties or function. We have recently succeeded in purifying this protein to greater than 80% homogeneity and have done preliminary studies of its physical and biochemical properties. The purified protein exists as variable sized oligomers which can bind saturably to trypsin sensitive sites on red cell inside-out vesicles. Moreover, the purified protein does not bind to red cell right-side out vesicles or to inside-out vesicles which have not been depleted of endogenous band 4.2 The goal of this proposal is to elucidate the physical and biochemical properties of band 4.2 in normal red cells and in red cells from individuals with inherited hemolytic anemias including hereditary spherocytosis (HS) and hereditary eliptocytosis (HE) Our efforts will focus on the following areas, 1) We will determine the membrane binding site or sites for band 4.2. Preliminary studies suggest that band 3 is one but not the only binding site for band 4.2 Studies using selective proteolysis will be used to localize the band 4.2 binding site within the primary structure of band 3. 2) We will determine whether band 4.2 associates with other membrane components such as membrane lipids, glycophorin, ankyrin and membrane skeletal proteins such as band 4.1 3) we will attempt to discover the function of band 4.2 by testing its effects on such features as band 3 catalysed anion transport, ankyrin binding to band 3, and band 4.1-membrane associations. 4) We will examine selected aspects of band 4.2 function and associations in red cells of individuals with inherited hemolytic anemias including HS and HE and 5) we will determine whether analogues of band 4.2 are present in non-erythroid cells and if so whether their distribution within certain cells or in relation to cellular structures can provide clues to their function. By elucidating the function of this major membrane constituent these studies will further our understanding of red cell biochemistry and physiology. Moreover, since band 4.2 deficiency or lack has been reported in association with several diseases such as hereditary spherocytosis and obstructive jaundice, our work may have significant implications for understanding the pathophysiology of these and related disorders.